PROJECT SUMMARY/ABSTRACT Despite accounting for significant mortality and morbidity in many disease states1?3, including primary hematologic disorders, cancer4, and sepsis5,6, our knowledge of bleeding and thrombotic complications remains far from complete due to a lack of tools. During blood clot formation, platelets undergo muscle-like contraction with nascent fibrin polymers to dynamically contract and stabilize the clot to stem hemorrhage. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics remains largely unknown yet clinically relevant, as overly softened and stiffened clots are associated with bleeding7 and thrombotic disorders8, respectively. Bulk assays are insufficient for these studies as individual platelets exhibit highly variable behavior that depends on the local clotting biochemical concentration and mechanical stiffness. Here, a newly developed high-throughput platelet contraction cytometer now enables parallel measurements of single platelet contraction forces in varied microenvironments. In addition to enabling detailed studies of individual platelet behavior, our preliminary data from patients suggests that the platelet contraction cytometer may represent an entirely new category of diagnostic that identifies a link between aberrant platelet contractile force and bleeding. As between 30-60% of patients with bleeding remain undiagnosed, this may represent a significant and impactful avenue to better understand and identify aberrant hemostasis. We found that highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a small sample patients with undiagnosed bleeding disorders. Similarly, we found correlations with active bruising and platelet contractile forces in the context of patients with immune thrombocytopenia (ITP). Moreover, platelet contraction cytometery appears to be independent of the known markers of platelet activation (phosphatidylserine, P-selectin, and PAC-1). The research objective of this NIBIB Trailblazer Award for New and Early Stage Investigators (R21) is to investigate a novel hypothesis: that the contractile force of platelets independently predicts bleeding. We will first define the healthy platelet contractile response to both collagen and fibrin(ogen) over the range of physiological stiffnesses present in a clot. We hypothesize that these various ligands will elicit different force responses from platelets, and we will analyze these responses and look for microenvironmental conditions that synergize to produce a highly contractile platelet phenotype, which we have previously found to have diagnostic potential. In addition, we will analyze the platelet contractile forces from: healthy volunteers, undiagnosed patients with symptomatic bleeding, and patients with immune thrombocytopenic purpura to test the hypothesis that impaired platelet contractile force will correlate with bleeding symptoms.